The long-term goal of this grant is to deduce general rules for how protein kinase signal transduction pathways maintain fidelity in signaling. Research over the past 10 years in this laboratory and other laboratories indicates that specificity in signaling occurs through a rapidly reversible regulated assembly of specific protein complexes in subcompartinents of the cell. The hypothesis guiding this study is that domains on signaling proteins mediate assembly into specific complexes via interaction with relatively short sequence motifs on target proteins. Additional fidelity is derived by the specificity of protein kinase catalytic domains for unique substrate motifs. Thus, subtle differences in kinases either within motifs or in residues that form the pockets of domains can explain their abilities to assemble into distinct complexes and phosphorylate distinct proteins in vivo. We have developed a peptide library screening approach that allows one to rapidly deduce optimal motifs for binding to protein interaction domains or to deduce optimal sequences for phosphorylation by protein kinases. The aims of the research are. 1) To use this approach to deduce optimal binding motifs for protein binding domains and protein kinases implicated in cell growth and cell survival pathways; 2) To isolate novel domains that bind phosphoSer (or phosphoThr) sites phosphorylated by specific protein kinases using a novel approach that we have developed; 3) To apply a new peptide library approach for determining substrates of proteases involved in cell growth and survival pathways; 4) To ascertain the structural basis for sequence-specific binding to the domains explored in aims 1 and 2; 5) To develop a web-based program that utilizes the information from the peptide library scans to predict binding sites and phosphorylation sites on proteins based on primary sequences.